Ethanol is one of the most widely consumed chemicals worldwide and has long been suspected of potentiating carcinogenesis by other chemicals. Numerous animal experiments have focused on this point. However, ethanol itself is not regarded as mutagenic. Acetaldehyde, the initial metabolic product of ethanol, is, in contrast, found to be a mutagen and weak carcinogen. The chemical basis for these and other observed biological effects is obscure, since ethanol does not bind to nucleic acids, while acetaldehyde forms a transient, reversible derivative on exocyclic amino groups. This laboratory has now isolated and characterized mixed acetals resulting from a cooperative reaction of acetaldehyde and ethanol. The new type of stable product forms when exocyclic amino groups of nucleosides react rapidly under gentle conditions (22o, pH6-8) with acetaldehyde and other alcohols. It has recently been found that the addition of acetaldehyde can be avoided, if alcohol dehydrogenase and its cofactor (NAD) are added, thus simulating biological conditions. This reaction represents a unique case in which the ingested chemical, e.g., ethanol, only reacts in the presence of its metabolite. The structure of the product is -NH-CH-(CH3)-O-C2H5. Formation and persistence of such mixed acetals may play a role inhuman carcinogenesis, particularly since modifications on the N4 of C are generally mutagenic. The reaction will be studied in detail (mechanism, kinetics, stability, etc.). Products will be identified and characterized by mass spectra, nmr, and optical methods. Reactions will be performed at low ethanol concentration with [3H] labeled deoxynucleosides, and with polynucleotides and [14C] labeled ethanol, all under physiological conditions (pH, temperature, ionic strength). Modified dNTPs will be prepared by direct reaction and used to study recognition and fidelity by DNA polymerases. The triphosphorylation of modified deoxynucleosides would be an alternative procedure to obtain the very pure NTPs needed if mutation frequency is low. Polymerases of differing origin will be tested using derivative-containing synthetic deoxy polymers as templates. The method of choice is site- directed mutagenesis experiments, with established protocols for oligonucleotide sequences from M13 phage.